Microfluidic Devices for Experiments on Bacterial Aerotaxis and for High-Resolution Imaging of Motile Bacteria

Abstract

Bacterial aerotaxis, directional motion of bacteria in gradients of oxygen concentration (often called oxygen taxis), was discovered 130 years ago and has since then been reported in a variety of different bacteria. In spite of numerous studies, quantitative results an bacterial aerotaxis have always been difficult to obtain because of the lack of appropriate instrumentation. Here, we built and characterized an experimental setup consisting of a computer-controlled 3-channel gas mixer and a two-layer microfluidic device, generating stable linear gradients of oxygen concentration, [O2], across a long test channel, with [O2] as small as 0.25% (corresponding to microaerobic conditions) in the middle. The test channel was continuously perfused with a suspension of E. coli cells and distributions of cells across the channel near its outlet were measured, with data on aerotaxis of ∼105 different cells collected within <1 hour. Extensive series of experiments on aerotaxis of E. coli cells were performed in linear gradients of [O2] with different slopes and mean concentrations. The experiments indicated that, in contrast to what was believed before, at [O2] up to ∼13%, E. coli always prefer highest accessible [O2].We also built and characterized microfluidic bacterial culture devices (chemostats) with the culture chamber depths adjustable between ∼2 and <0.5 μm with a resolution of ∼20 nm. By changing the chamber depth, motile bacterial cells of different species (B. subtilis, E. coli, C. crescentus) were gently immobilized for high-resolution fluorescence imaging and released after the imaging was completed. The technique also enabled culturing motile C. crescentus cells in semi-permeable microchambers, thus making it possible to generate C. crescentus colonies of exceptionally high density and observe the behavior of cells in these colonies.